The present invention relates to a membrane which is intended particularly, but not exclusively, for use in a sensor, to a sensor incorporating such a membrane, and to a detection method utilising the membrane. The membranes of the invention are intended most particularly (but again not exclusively) for use in chemical and biosensors, e.g. for determining the amount of lactate in whole blood, (this being a measure of the oxygenated state of tissue).
Biosensors are used for detecting the presence and/or amount of a selected component in a sample, e.g. blood. It is well known that biosensors comprise a combined detection arrangement of a biolayer such as an enzyme and a transducer. Certain types of biosensors have a membrane structure which comprises one or more membranes and which (in use of the biosensor) separates the detection arrangement from the sample being analysed. The membrane structure may contain a biological component (e.g. an enzyme) but usually separates the biological component from the sample. In the case of an enzyme there is a reaction with the species of interest to produce a product which is detected by the transducer; this gives one type of arrangement for xe2x80x9cindirectxe2x80x9d determination of the species of interest.
The membrane structure of the biosensor will generally include an outer membrane of a synthetic polymeric material which has been modified to produce a membrane which is permeable to the species of interest (or more permeable than a similar membrane produced from polymeric material which has not been modified). If the biosensor is to be used for operation in whole blood then the outer membrane (of the membrane structure) which comes into contact with the blood has to meet high demands on biocompatibility since sample pre-treatment preferably does not take place. The membrane surface will be a target for plasma proteins which will begin to adsorb rapidly after the initial contact with blood. Initial protein adsorption will be followed by complement activation, adhesion of cells and cell components and fibrin clot formation.
Operation in whole blood also puts demands on the linear range of the sensor which has to be much wider when there is no dilution of the sample prior to analysis.
According to a first aspect of the present invention there is provided a membrane comprised of a synthetic polymeric material incorporating a surface active agent.
By xe2x80x9csurface active agentxe2x80x9d we also mean xe2x80x9csurfactantsxe2x80x9d and the terms may be used interchangeably.
The invention has been based on our finding that the incorporation of a surface active agent in a synthetic polymeric material renders membranes, produced from that material permeable (or more permeable) by mass transport to a species of interest whilst providing membranes which have good blood compatibility and which may be used to provide a responsexe2x80x94linearising diffusion barrier. This is particularly the case for enzymes which have a reaction with the species of interest which has a rate that is not linear with respect to substrate concentrations when these are high (typically well above the enzyme Michaeli""s constant).
The membranes may be rendered permeable to uncharged species or to charged species and may therefore be used in biosensors for determining the presence and/or amount of such charged species, e.g. the concentration of lactate present in a sample.
Membranes in accordance with the invention have the significant advantage of being blood compatible and may therefore be used in chemical sensors and biosensors for determining the amount of a species in whole blood. The membranes are thus particularly suitable for use in biosensors for determining the amount of lactate in whole blood. The membranes of the invention may however be used for other purposes, e.g. dialysis and as permeable covering layers on other enzyme and even non-enzyme electrodes. Moreover the membranes have utility in sensors for determining uncharged species, e.g. glucose and in chemical sensors which have no biological component.
It is a further advantage of the membranes of the invention that they provide a linearising diffusion barrier for the species of interest, i.e. the amount of the species present in a sample which is able to diffuse across the barrier is proportional to the amount of that species in the sample, but the concentration achieved after the barrier is crossed is thereby lowered and within the range for which the chemical or biosensor has a linear signal output.
Preferably the synthetic polymeric material is poly(vinylchloride). Preferably the poly(vinylchloride) will have a molecular weight (Mw) in the range 80,000 to 250,000, more preferably 150,000 to 250,000, e.g. about 200,000.
The amount of the surface active agent present in the membrane is generally at least 1% by weight of the synthetic polymer, e.g. more preferably at least 2%, even more preferably at least 3% on the same basis. Generally the amount of surface active agent is less than 250% by weight of the synthetic polymer. If the membrane is to be used in a sensor as a linearising diffusion barrier then the amount of surface active agent will depend on the intended use of the sensor. For some applications (e.g. use of the sensor under continuous flow conditions) it may be desirable for the amount of surface active agent to be at least 50% by weight of the synthetic polymer. For other applications the amount of surface active agent may be less than 50% (w/w) of the synthetic polymer, e.g. less than 30% or even less than 20% on the same basis. For dialysis membrane function, high mass transport (permeability) is required and higher amounts of surface active agent are demanded, typically in the range 100% to 200% (more preferably 150% to 200%) (w/w) of the synthetic polymer.
The surface active agent is preferably a non-ionic surface active agent. Such non-ionic surface active agents are particularly useful for avoiding specific ionic interactions with diffusing solute. Alternatively the surface active agent may be a cationic or anionic surfactant.
A variety of non-ionic surface active agents may be used. Surface active agents which are useful in the present invention include compounds comprised of or incorporating polyoxyalkylene residues. The alkylene oxide may for example be ethylene oxide and/or propylene oxide. Examples of such surface active agents include compounds of the formula (I) 
Typically the molecular weight of compound (I) will be in the range 500 to 800, e.g. from 600 to 700. A particularly suitable product of the formula (I) has a molecular weight of about 648 and is available under the name Triton X-100.
Further examples of surface active agents comprising poly(alkylene oxide) residues are block copolymers of ethylene oxide and propylene oxide. Suitable examples of such copolymers for use as surfactants have a molecular weight of 5000 to 10000, more preferably 7000 to 10000.
The block copolymer may be of the following general formula (I). 
The block copolymer of ethylene oxide and propylene oxide may for preference have an ethylene oxide content of at least 75%, preferably about 80%, by weight of the surfactant.
Examples of preferred surfactants comprised of block copolymers of ethylene oxide and propylene oxide are available under the name PLURONIC. A particularly suitable example of such a product is available under the name Pluronic F-68 and has a molecular weight of about 8400.
If the surface active agent comprises a block copolymer of ethylene oxide and propylene oxide then it (i.e. the surface active agent) may comprise, for example, 4.5 to 100% by weight of the synthetic polymeric material, e.g. 6% to 100%, more preferably 10% to 90%, even more preferably 40% to 70%, and even more preferably 50% to 60% by weight on the same basis.
A further surface active agent which may be used is L-xcex1-phosphatidyl choline dipalmitoyl (C40H80NO8P) (Mw=734). This surface active agent occurs naturally as a pulmonary surfactant and has advantages from the point of view of biocompatibility. The inclusion of surfactants has generally led to improved haemocompatability in comparison with non-surfactant PVC membranes.
It is also possible to use analogues of surface active agents and such analogues are to be understood to be covered by the terms surface active agent as used herein. A particularly preferred analogue is bis(2-ethylhexyl) hydrogen phosphate which is of the formula 
The above compound is referred to herein as BEP.
An important feature of a membrane comprised to PVC incorporating BEHHP is that membrane permeability can be made to be dependent on both buffer composition and pH. At pH 7 in either phosphate or carboxylic acid based buffers the PVC (BEHHP) membrane exhibits high permeability characteristics similar to those observed for PVC modified with either Tween 80 or Triton X-100. However, when interfaced with carboxylic acid based buffers (e.g. succinate buffer), at a pH where the acid buffer is predominantly in the neutral form (i.e. below it""s pKa value), the PVC (BEHHP) will tend to switch to a low permeability state. PVC (BEHHP) could therefore be useful as a pH sensitive solute gating membrane.
Preferred membranes for use in a lactate sensor (referring to lactate determination in undiluted whole blood) comprise polyvinylchloride incorporating 10 to 45% (e.g. 18% to 45%) (w/w) of the non-ionic surface active agent Triton X-100 or 4.5-100% (w/w) of Pluronic F-68 or 1-3% (w/w) of L-xcex1-phosphatidyl choline dipalmitoyl, the percentages being based on the weight of the poly(vinyl chloride). These values are particularly preferred for sensors which have an inner selective membrane (see later).
If the membrane is to be used for dialysis, the amount of surface active agent is preferably 150% to 200% (w/w/).
The membranes will typically have a thickness of 0.1 to 200 microns.
Membranes in accordance with the invention may be produced by conventional casting techniques. Alternatively the membrane may be produced by a xe2x80x9cspin coatingxe2x80x9d technique in which a solution of the polymer in a volatile solvent is applied to a flat support surface which is then rotated (usually about a vertical axis) at a speed which causes the solution to be evenly distributed on the support and the solvent to be evaporated so as to produce a thin polymeric membrane of uniform thickness. The spin coating procedure allows membranes having a uniform thickness of 2 to 10 (e.g. 4 to 6) microns to be produced very quickly, e.g. from ten seconds to two minutes. The thickness of the membranes produced by the spin coating technique may also be readily controlled. The solution from which the membrane is formed by spin coating will generally have a concentration of 1 to 15% by weight, e.g. 2 to 10% by weight. To form the membrane, the polymer solution is applied on the axis about which the support is to be spun. Rotational speeds of 250 to 1500 rpm will generally be suitable. The support will generally be spun for up to two minutes, e.g. 90 seconds, to produce the desired membrane. A suitable apparatus for producing membranes by spin coating is a Photoresist Spinner as used for producing layers of photoresist materials. In all cases, a continuous membrane is produced in which the miscible surface active agent is distributed.
According to a second aspect of the present invention there is provided a sensor device comprising detecting means for detecting the amount of a species of interest in a sample and a membrane in accordance with the first aspect of the invention providing both a barrier function and a biocompatible interface function between the detecting means and the sample.
According to a third aspect of the present invention there is provided a method of determining the amount of a selected component in a sample the method comprising using a sensor device which incorporates means for detecting the amount of the component in a sample and a membrane in accordance with the first aspect of the invention, said membrane being located in contact with the sample and providing both a barrier function and a biocompatible interface function between the sample and the detection means.
The sensor device of the second aspect of the invention is preferably a biosensor but may be another form of sensor device.
The detecting system preferably comprises an electrochemical electrode system but may alternatively be in some other form such as a spectophotometric or optical system where physical contact with the sample is required. Where an electrochemical electrode system is used, it is preferably of the non-potentiometric type, examples of which include amperometric, galvanic, photo-galvanic and coulometric types. Most preferably the sensor device is a biosensor in which the detecting means comprises an amperometric electrode.
The method of the third aspect of the invention is particularly suitable for the determination of lactate in whole blood using an electrode detection system. In such a method, the lactate may be detected indirectly. Thus, for example, after diffusion through the membrane the lactate may interact with an enzyme on the electrode side of the membrane such that the interaction results in the production or consumption of a species which can be directly detected electrochemically. The enzyme may be immobilised in a layer provided between the membrane and the electrode. This layer may, for example, comprise the enzyme cross-linked with bovine serum albumin in a glutaraldehyde matrix. The enzyme may be entrapped in the membrane which may directly contact the sample or have an additional membrane containing surface active agent interposed between the enzyme containing membrane and sample.
The enzyme may, for example, be one which leads to the production of hydrogen peroxide which may then be detected. A suitable enzyme is an oxidase, e.g. lactate oxidase.
The enzyme layer may, if desired, incorporate catalase.
In addition to being permeable to lactate, the membrane of the invention may additionally be permeable to other species which could be interferents in the analysis of a particular sample using an enzyme electrode. In the case of blood, electroactive interferents include ascorbate, urate and acetaminophen (paracetamol), all of which will diffuse through the membrane of the invention. Sensors in accordance with the invention will therefore generally additionally include a further membrane (between the enzyme and the electrode) to provide selectivity against the interferents. Most preferably such a selective membrane is comprised of a sulphonated poly(ether ether sulphone)/(poly(ether sulphone) polymer (SPEES-PES) or cellulose acetate. Both without such a selective membrane or a selective biolayer (e.g. enzyme), direct electrochemical or other detection of electrode-active species is possible without external electrode fouling.
The sensor may be employed under continuous flow conditions, i.e. the sample to be analysed is supplied as a continuous flow to the sensor.
This method is particularly applicable to the determination of lactate in whole blood using a sensor device in which the detecting means comprises an enzyme electrode system of the type disclosed above, e.g. lactate oxide immobilised in a glutaraldehyde matrix. In monitoring the amount of lactate in whole blood under continuous flow conditions the blood may be treated with an anti-coagulation agent and then passed through a flow-through measurement cell. The necessary flow may be provided by a peristaltic pump.
Conveniently, the blood to be sampled is passed in one direction along the inner lumen of a double lumen catheter whilst the anti-coagulation agent is passed in the opposite direction through the outer lumen for admixture with the blood as it enters the inner lumen.
The anti-coagulant should be present at a sufficient concentration to prevent coagulation. Preferably the anti-coagulation agent is heparin.
The flow rate of the anti-coagulation agent may be at least equal to that of the blood. Thus, for example, the blood may provide 20 to 50%, eg. 30% of the total volume of mixed blood and anti-coagulant.
In use of a device in accordance with the second aspect of the invention it is possible (and may possibly be desirable) that there will be some continuous leakage of surface active agent from the membrane surface in contact with the sample. In fact, continuous loss of surface active agent and its surface replacement may eliminate fouling by resulting in a self-regenerating and therefore self-cleaning surface. In fact, the degree of fouling observed microscopically associated with devices incorporating membranes in accordance with the first aspect of the present invention is amongst the lowest for any known membrane or polymer material that has been exposed to blood.
It may therefore be desirable to provide an additional source of surface agent to replenish that lost from the membrane. Thus for example, a reservoir of surface active agent may be provided around an extended area of the membrane (i.e. on one or both sides of the membrane extended beyond the detection surface of the sensor). Alternatively a source of surface active agent may be pre-loaded into the extended side of the membrane remote from the detection means and from the part of the membrane exposed to sample and serve to supply surface active agent through the membrane to replace that lost across the part of the membrane in contact with the sample.